Biologically active synthetic thyrotropin and cloned gene for producing same

ABSTRACT

Substantially pure recombinant TSH has been prepared from a clone comprising complete nucleotide sequence for the expression of the TSH. Diagnostic and therapeutic applications of the synthetic TSH are described.

RELATED APPLICATIONS

This application is a continuation of application Ser. No. 11/543,498filed Oct. 5, 2006, which is a is a continuation of application Ser. No.11/376,778 filed Mar. 15, 2006, now abandoned, which is a continuationof application Ser. No. 11/148,604 filed Jun. 9, 2005, now abandoned,which is a continuation of application Ser. No. 10/737,469 filed Dec.16, 2003, now abandoned, which is a continuation of application Ser. No.09/892,266 filed Jun. 27, 2001, now abandoned, which is a continuationof application Ser. No. 09/569,141 filed May 11, 2000, now U.S. Pat. No.6,284,491, which is a continuation of application Ser. No. 08/310,923filed Sep. 22, 1994, now U.S. Pat. No. 6,117,991, which is acontinuation of application Ser. No. 08/110,639 filed Aug. 23, 1993, nowabandoned, which is a continuation of application Ser. No. 07/882,231filed May 8, 1992, now abandoned, which is a continuation of applicationSer. No. 07/295,934 filed Jan. 11, 1989, now abandoned, all of which arehereby expressly incorporated by reference in their entireties.

FIELD OF THE INVENTION

The present invention is related generally to the isolation andcharacterization of new genes and proteins. More particularly, thepresent invention is related to providing isolated, substantially pure,biologically active human thyrotropin (hTSH) synthesized by a clonedgene.

BACKGROUND OF THE INVENTION

Thyrotropin (TSH) is a pituitary peptide hormone which regulatesimportant body functions. However, heretofore there was no stable,reliable and economic means of synthesizing this important hormone.

SUMMARY OF THE INVENTION

It is, therefore, an object of the present invention to providebiologically active, synthetic human thyrotropin in substantially pure,isolated form.

It is another object of the present invention to provide a cloned genewhich directs the expression of biologically active human thyrotropin ina suitable vector.

It is a further object of the present invention to provide an assay kitfor measuring thyroid-stimulating hormone as well as other thyrotropinsubstances such as thyroid-stimulating immunoglobulins and the like.

It is a further object of the present invention to provide a method ofdiagnosing and treating human thyroid cancer.

Other objects and advantages will become evident from the followingdetailed description of the invention.

BRIEF DESCRIPTION OF THE DRAWINGS

These and other objects, features and many of the attendant advantagesof the invention will be better understood upon a reading of thefollowing detailed description when considered in connection with theaccompanying drawings wherein:

FIG. 1 shows schematic construction of the expression vectors. pSV2.Gand pAV2 are pBR322 derived plasmids with the origin of replication(ori) and ampicillin resistance gene (amp r) as shown. pSV2.G containsthe early promoter of SV40 upstream of the HindIII cloning site, rabbitβ-globin cDNA, and poly-adenylation site/intron of SV40. pAV2 has theentire adenovirus-associated virus genome (4.7 kb) with its threepromoters P5, P19, P40, and polyadenylation site. The HindIII cloningsite is downstream of the P40 promoter. Human TSHβ and hCGα was insertedinto the HindIII site of either plasmid, forming pAV2-hTSHβ, pAV-hCGα,pSV2.GhTSHβ, and pSV2.G-hCGα.

FIG. 2 shows Northern blot analysis of transfected 293 and COS cells.Total cellular RNA was separated on a 1% agarose-formaldehyde gel andtransferred to a nylon membrane. Forty micrograms of total RNA from acontrol of transfected cell culture were applied to each lane. Human CGαand hTSHβ were abbreviated α and β in construct names and in otherfigures. Cells that were not transfected are labeled control. Cellstransfected with a calcium phosphate precipitate lacking DNA are labeledmock. Lanes 1-4 are total RNAs derived from 293 cells; lanes 5-9 areRNAs derived from COS cells. The migration position of an RNA standardin kilobases and hTSHβ mRNA from human pituitary is shown to the left ofthe autoradiograph. Below the autoradiograph is a simplified version ofFIG. 1 showing the pAV2 and pSV2.G plasmid as a single line, thepromoters as blackened circles, the 2.0 kb hTSHβ genomic fragment as abox containing two exons (blackened regions) and known polyadenylationsignal-site sequences as open arrowheads. Below each construct, pAV2-βand pSV2.G-β, is the predicted RNA initiating at the specified promoter,and splicing as shown. Solid arrowheads, poly(A) tails. Predicted sizein kilobases (kb) is shown to the right of each mRNA species.

FIG. 3 shows the results of gel chromatography. Cell medium from 293cells transfected with pAV2-hCGα/pAV2-hTSHβ/pVARNA was chromatographedon a Sephadex G-200 fine column. In addition, standard preparations ofhTSH, hCGα, and hTSHβ (described in the text) were chromatographed onthe same column. RIA of human α and TSHβ and IRMA for hTSH was done oneach 1.5-ml fraction. Elution position of bovine thyroglobulin (void,V_(o)), BSA (67,700 (67 k)) and ovalbumin (45,000 (45 k)) is marked, aswell as those of the standard preparations of hTSH, hCGα, and hTSHβ.

FIG. 4 shows the results of human TSH IRMA. A highly sensitive andspecific hTSH IRMA was performed on two pituitary hTSH standards. WorldHealth Organization 80/558 (WHO STD) and NIH I-6 (1-6 STD), and themedium from 293 cells after transfection with pAV2-hCGα, pAV2-hTSHβ, andpVARNA (pAV2α/β/pVARNA). A logit transformation of assay binding wasplotted vs. arbitrary units of sample volume added to the assay.

FIG. 5 shows the results of in vitro bioassay of hTSH in rat thyroidcells. The human pituitary TSH standards and medium from transfected 293cells used in this assay are defined in the legend to FIG. 4. This invitro bioassay measures TSH stimulated ¹²⁵I uptake into rat thyroidcells (FRTL5). Iodide trapping by pituitary standards and medium from atransfected culture are normalized to TSH immunoactivity in an IRMA.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT

The above and various other objects and advantages of the presentinvention are achieved by the cloning of complete nucleotide sequencewhich directs the synthesis of biologically active human thyrotropin ina suitable expression vector and isolating substantially pure form ofthe synthesized hormone.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Although any methods andmaterials similar or equivalent to those described herein can be used inthe practice or testing of the present invention, the preferred methodsand materials are now described. All publications mentioned hereunderare incorporated herein by reference. Unless mentioned otherwise, thetechniques employed herein are standard methodologies well known to oneof ordinary skill in the art.

The term “substantially pure” as used herein means as pure as can beobtained by employing standard conventional purification techniquesknown in the art.

The term “biologically active” as used herein means that the recombinanthormone, even though not identical in physical or chemical structure orcomposition as the naturally occurring hormone, yet is functionallyequivalent thereto.

Materials and Methods

Materials

Restriction and modifying enzymes were obtained from Bethesda ResearchLaboratories (Gaithersburg, Md.) and Pharmacia (Piscataway, N.J.). ³²Pand ³⁵S compounds were purchased from both DuPont New England Nuclear(Boston, Mass.) and Amersham/Searle Corporation (Arlington Heights,Ill.). Gene Screen and Gene Screen Plus membranes (New England Nuclear)were used in all DNA and RNA transfer procedures. A transformationcompetent strain of Escherichia coli, HB101, was obtained from BethesdaResearch Laboratories and used in all transformations. Oligonucleotideswere purchased from the Midland Certified Reagent Company (Midland,Tex.). Cloning and propagation of DNA was done in accordance with NIHguidelines. Sephadex G-200 fine and concanavalin A-Sepharose wereobtained from Pharmacia Fine Chemicals. α-Methyl glucoside and α-methylmannoside were purchased from Sigma (St. Louis, Mo.). Human TSH, hCGα,and hTSHβ were provided by the NIDDK National Hormone and PituitaryProgram (Bethesda, Md.). Protein standards were purchased from Sigma orPierce Chemical Co. (Rockford, Ill.).

Genomic Screening

Independent recombinant phage clones (1×10⁶) of an EMBL3 human genomicleukocyte library were screened for the presence of human TSHβ using aradiolabeled mouse TSHβ cDNA obtained from W. Chin, Brigham and Women'sHospital, Harvard Medical School, Boston, Mass., and two separate cloneswere identified. A 34 base oligonucleotide, with the same sequence asthe first 34 bases of the 5′-untranslated sequence of bovine TSHβ cDNA,was 5′-end labeled with [τ-³²P]ATP to a specific activity of 5-8×10⁶cpm/picomol using polynucleotide kinase; mouse TSHβ cDNA was [α-³²P]dCTP labeled with a random primer to a specific activity of 1-5×10⁹cpm/μg. Both were used to probe Southern blots of restriction digests ofone clone.

Subcloning and Sequencing

Genomic fragments were subcloned into pUC18 and mp13 to facilitaterestriction mapping and sequencing using the dideoxy chain terminationmethod of Sanger (Sanger et al. 1977 Proc Natl Acad Sci USA74:5463-5467).

Expression Vectors

A 621 bp hCGα cDNA (obtained from J. Fiddes, California BiotechnologyInc., Palo Alto, Calif.) was inserted downstream of the early promoterof SV40 in pSV2.G (obtained from B. Howard, NIH, Bethesda, Md.) (Gormanet al. 1982, Mol Cell Biol 2:1044-1051) or the P40 promoter ofadeno-associated virus in pAV2 (Laughlin et al. 1983, Gene 23:65-73) atthe HindIII site forming pSV2.G-hCGα and pAV2-hCGα (FIG. 1). HindIIIIlinkers were ligated to a 2.0 kb PvuII fragment of the hTSHβ genecontaining 277 bp of 5′-intron, both coding exons, a 450 bp intron, andapproximately 800 bp of 3′-flanking DNA. It was inserted into the sameHindIII sites as hCGα forming pSV2.G-hTSHβ and pAV2-hTSHβ (FIG. 1). Allplasmids were subjected to multiple restriction enzyme digestions toconfirm the presence of only one insert in the proper orientation.

Cell Culture

Adenovirus transformed human embryonal kidney cells (293 cells) and SV40transformed monkey kidney cells (COS cells) were grown in a modifiedminimal essential (MEM) and Dulbecco's modified Eagle's medium,respectively. Both media were supplemented with 10% fetal bovine serum,4.4 mM L-glutamine, 100 U/ml penicillin, 100 μg/ml streptomycin, and 250ng/ml amphotericin B. Twenty-four hours before transfection, the cellswere replated on 100-mm dishes at the same density (5×10⁵). On the dayof transfection fresh medium was added to the cells 4 h beforetransfection.

Transfection

All transfections were performed using the calcium phosphateprecipitation method (Graham et al. 1973, Virology 52:456-467). Theprecipitate was applied for 4 h, the cells were washed, and fresh mediumwas added. Total RNA was isolated according to the method of Cathala etal. 1983, DNA 2:329-335. The pAV2 plasmids were transfected into both293 and COS cells in Exp 1 and into only 293 cells in Exp 2. The pSV2.Gplasmids were only transfected into COS cells. When either the α- orβ-subunit was transfected alone into cells, 15 μg purified plasmid wereapplied to each plate. When both the α- and β-subunit werecotransfected, 9 μg each purified plasmid were applied to one-plate. Insome cases, cells were cotransfected with pVARNA. pVARNA consists of anadenovirus type 2 DNA HindIII B fragment containing the genes for VA_(x)and VA_(xx) inserted into the HindIII site of pBR322 (obtained fromKetner, Department of Biology, Johns Hopkins University, Baltimore,Md.). VA_(x) RNA stimulates translation by inhibiting phosphorylationand inactivation of the a subunit of eucaryotic initiation factor 2(Akusjarvi et al. 1987, Mol Cell Biol 7:549-551).

RNA Analysis, RIA, and IRMA

Northern blot analysis of total RNA from transfections was performedusing standard methods (Maniatis et al. 1982, Molecular Cloning, ed 1,Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y., p 202) andmanufacturer's specifications. Common human α-subunit RIA, hTSHβ-subunitRIA, and hTSH IRMA were performed in duplicate on the medium from eachtransfected culture (McBride et al. 1985 Clin Chem 31:1865-1867;Kourides et al. 1974 Endocrinology 94:1411-1421). The sensitivities ofthe assays were less than 0.03 ng/ml, less than 0.03 ng/ml, and lessthan 0.06 ng/ml, respectively. Cross-reactivity between thecorresponding subunit and hTSH, at the measured concentration, was lessthan 5% in the common α and less than 2% in the hTSHβ RIA (Kourides etal., supra). In addition, at the measured free subunit concentrations,the hTSH IRMA exhibited less than 1% cross-reactivity (data not shown).

Gel and Lectin Affinity Chromatography

The apparent mol wt of hTSH synthesized in 293 cells was determined bygel chromatography on a 1.5×90-cm Sephadex G-200 fine column calibratedduring each chromatography run with five protein standards (bovinethyroglobulin, BSA, ovalbumin, bovine chymotrypsinogen A, and whalemyoglobin). The column was equilibrated and run at 4° C. in a buffercontaining 0.12 M sodium chloride, 0.1 M borate, and 0.02% (wt/vol)sodium azide, pH 7.4. Two milliliters of fresh MEM medium containing 100μUhTSH (WHO 80/558). 100 ng hCGα (CR-119), and 100 ng hTSHβ (APP-3929β)were applied during chromatography of standard preparations. The columnwas washed and then 2 ml MEM medium from 293 cells transfected withpAV2-hCGα/pAV2-hTSHβ/pVARNA were applied. Fractions of 1.5 ml werecollected at a flow rate of 6 ml/h.

The binding of hTSH synthesized in 293 cells to concanavalin A-Sepharosewas also determined using methods previously described (Gesundheit etal. 1987, J Biol Chem 262:5197-5203). Samples were applied to lectincolumn, and 2-ml fractions were collected at a flow rate of 10 ml/h.Human TSH IRMA was common human α- and hTSHβ-subunit RIAs were performedon fractions from gel and lectin chromatography. Recovery of hTSH andits free subunits was generally greater than 90% from chromatography.

TSH Bioassay

Thyrotropic bioactivity was measured as the ability to stimulate theuptake of ¹²⁵I into rat thyroid cells (FRTL5) in accordance with theprocedure of Dahlberg et al. 1987 J Clin Invest 79:1388-1394. This assaymeasures human, rat, and bovine thyrotropin but not gonadotropins orfree α- or TSHβ-subunits. Sample determinations were performed induplicate and compared to two pituitary hTSH standards (World HealthOrganization 80/558 and National Institutes of Health 1-6). Results areexpressed as microunits per ml; one microunit of WHO 80/558 isequivalent to 0.09 ng of NIH 1-6 purified hTSH (unpublished data).

Statistics

Significant differences in immunoassay of cell media from variouscontrol and transfected cultures were determined using Student's t test.

RESULTS

Human TSHβ Gene

A 17 kb genomic fragment was isolated by screening 1×10⁶ recombinantphage clones. A restriction map (FIG. 1) was constructed using Southernblots of phage DNA hybridized with both a mouse TSHβ cDNA probe (lacking5′-untranslated sequences) and a 34 bp bovine 51-untranslated sequenceprobe. Two coding exons are separated by a 450 bp intron and thesequence is identical to the published partial sequence (Hayashizaki etal. 1985 FEBS Lett 188:394-400) (data not shown). However, the completecoding sequence was not heretofore known.

Transfection

Two experiments were performed to compare the level of mRNA and proteinproduction between the most active adeno-associated virus promoter, P40,and the early promoter of SV40.

Table 1 shows the RIA and IRMA assay results from these two experiments.Interestingly, the 293 cells synthesized small amounts of free α-subunit(control) whose levels were increased approximately 10-fold in atransfection with the calcium phosphate precipitate but without DNA(mock) (P<0.0005). While neither transfection with pAV2-hCGα nor pVARNAincreased α-production above the level of a mock transfection, thecombination increased free α-levels 3- to 5-fold (P<0.0005). Thusexogenous sources of the human α-subunit were clearly important inmediating this increase. The same pattern of pVARNA increasing proteinproduction was seen when pAV2-hTSHβ was transfected. 293 cells do notproduce hTSHβ so that the medium of cells exposed to a mock or pVARNAtransfection did not have measurable hTSHβ. Only the 293 cells exposedto pAV2-hTSHβ produced hTSH. When the β plasmid was transfected alonethe hTSH formed was due to combination with endogenous α. Cotransfectionwith both α- and β-plasmids, though, increased hTSH levels 1.5- to2-fold (P<0.05).

COS cells synthesized neither free α nor β but could synthesize hTSHβand hTSH when transfected with the appropriate plasmids. The levels ofprotein production were 10- to 100-fold less than in 293 cells and wereonly measurable when the pVARNA plasmid was also transfected. Regardlessof whether pAV2 or PSV2.G was used, protein levels were barely if at allmeasurable without the pVARNA plasmid.

FIG. 2 shows a Northern blot of total cellular RNA hybridized with themouse TSHβ cDNA probe. In 293 cells, hTSHβ message was not detected fromnontransfected (control) or from mock transfected cells. However, threeRNA species of 2.3 kb. 1.6 kb, and 650 bases were noted aftertransfection of pAV2-hTSHβ and pVARNA (lane 3). These three bands arethe same size as those predicted from pAV2-hTSHβ if transcription beganat all three adeno-associated viral promoters (FIG. 2). The mRNA of 650bases presumably represent a properly spliced hTSHβ message. Lane 4shows the same three bands but at lesser intensity when pAV2-hCGα,pAV2-hTSHβ, and pVARNA were cotransfected. This reduction in signalintensity seen in lane 4 may have been due to the reduction in theamount of pAV2-hTSHβ transfected from 15 μg to 9 μg.

Control and mock transfected COS cells also did not contain hTSHβmessage. When pSV2.G-hTSHβ was transfected (lanes 7-9), a major band of900 bases and a minor band of 3.0 kb were seen. Without being bound toany theory, it is postulated that the 900 base species could represent amRNA with the 277 bases of 5′-intron remaining, while the 3.0 kb speciescould represent read through of the hTSHβ poly-adenylation signal-siteand use of the polyadenylation signal-site of pSV2.G (See FIG. 2).

Specific human α mRNA transcripts of appropriate size analogous to thehTSHβ mRNA above were observed in cells transfected with pAV2-hCGα (datanot shown). Since the main object of this invention is proteinexpression, the relative contribution of human a mRNA from endogenousvs. exogenous (pAV2-hCGα) sources in 293 cells was not determined.However, the data suggest that the high level of free α-subunit observedafter transfection with pAV2-hCGα/pVARNA is most likely due to mRNA fromexogenous sources.

Gel and Lectin Affinity Chromatography

The apparent molecular weight of hTSH and its subunits synthesized in293 cells after transfection with pAV2-hCGα/pAV2-hTSHβ/pVARNA wasdetermined on a G-200 Sephadex column (FIG. 3). In addition, standardpreparations of hTSH, hCGα, and hTSHβ were chromatographed on the samecolumn. Internal protein standards had identical elution patternsbetween runs as determined by optical density at 280 nm. In each case,the apparent mol wt of synthetic hTSH and its subunits was larger thanits corresponding standard. Specifically, synthetic hTSH displayed anapparent mol wt of 45.000 and was larger than standard pituitary hTSH(apparent M_(r)=40,000). This clearly indicates that the recombinant TSHis not constitutively identical to the natural product. The human α andhTSHβ from transfection coeluted with the hTSH pituitary standard, andboth were larger than their respective standard subunit preparation.However, in the case of free human α-subunit, the relative contributionto this chromatography pattern of endogenous α as compared to exogenousα from pAV2-hCGα cannot be determined.

The binding pattern to concanavalin A-Sepharose of synthetic hTSH from293 cells as, compared to standard human pituitary hTSH is shown inTable 2. Synthetic hTSH was glycosylated as indicated by completebinding to concanavalin A-Sepharose. The different elution pattern ofstandard vs. synthetic hTSH from the lectin columns is indicative of adifference at least in carbohydrate structure, again showing that therecombinant TSH (rTSH) is distinctly different from the naturallyoccurring TSH.

Immunoactivity and Bioactivity

FIG. 4 shows that the hTSH produced in cell culture wasindistinguishable from two pituitary hTSH standards in an assayinvolving two antibodies directed at different epitopes of the hTSHheterodimer (McBride et al., supra). The slopes were parallel over theentire range of values. FIG. 5 shows the same hTSH in a ¹²⁵I trapping invitro TSH bioassay compared to the same pituitary hTSH standards. The invitro bioassay of standard pituitary hTSH or, the cell culture productfrom 293 cells (pAV2-hCGα/pAV2-hTSHβ/pVARNA) was normalized toimmunoreactivity in a hTSH immunoradiometric assay (IRMA) assay. Thedose response, and ED₅₀ of the standards and cell culture product wereidentical. In addition the cell culture product from COS cells(pAV2-hCGα/pSV2β/pVARNA) was biologically active although the lowerlevel of expression prevented determination of a dose response curve.

In summary, a 17 kb genomic fragment of hTSHβ has been isolated and bothcoding exons of this gene produced hTSHβ and hTSH in a transientexpression assay. This is the first report of TSH from any speciesproduced by gene transfection in cell culture. The expression vectors ofhTSHβ included only the two coding exons, and not the 5′-untranslatedexon of the gene (Wondisford et al. Mol Endo 2(1):32-39, 1988).

Transient expression after gene transfection was used to test both theearly promoter of SV40 or the P40 promoter of adeno-associated virus.The early promoter in COS cells produced more mRNA than the P40 promoterin 293 cells regardless of whether pVARNA was cotransfected. However,pVARNA clearly increased mRNA levels in either vector system. Thissuggests that in addition to increasing the rate of translation, pVARNAmust either increase transcriptional rate, RNA transport, or stability.

While the pSV2.G-hTSHβ expression vector produced higher levels of hTSHβmRNA than pAV2-hTSHβ, this mRNA was about 250 bases larger than thatfound in the human pituitary. The 450 bp intron was certainly splicedout since this intron in the mature message would have prevented hTSHβprotein synthesis. Also, an mRNA of appropriate size was produced bypAV2-hTSHβ indicating that the polyadenylation site in the fragment mustbe active. Thus, the most likely reason for a larger hTSHβ mRNA in COScells was the lack of splicing of a 277 bp intron fragment upstream ofthe first coding exon. Eighteen base pairs downstream of thetranscriptional start site of the P40 promoter is a consensus splicedonor site which could explain why the 277 bp intron fragment would bespliced out in the adeno-associated virus vector.

The plasmid, pVARNA, increased protein production in either vectorsystem, but the P40 promoter in 293 cells led to expression of between10- to 100-fold more protein than the early promoter of SV40 whencotransfected with pVARNA. This is most likely due to an increasedtranslational rate mediated by pVARNA as has been previouslydemonstrated for expression of other mRNAs (Akusjarvi et al. 1987 MolCell Biol 7:549-551). Of course, the possibility that the larger mRNAfrom pSV2.G-hTSHβ contributed to the lower protein levels from COS cellscannot be excluded

The hTSH produced in cell culture was functionally indistinguishablefrom two pituitary hTSH standards in both a highly specific IRMA and invitro bioassay. It should be noted, however, that the synthetic hTSH ofthe present invention was larger in size than standard pituitary hTSH ongel chromatography. Although it was glycosylated as indicated bycomplete binding to concanavalin A, it displayed a somewhat differentpattern on lectin chromatography as compared to a standard hTSHpreparation. The larger mol wt of these synthetic glycoproteins ascompared to pituitary standards is most likely due to an alteredglycosylation pattern such as more sialylation. In the case of hTSH,this might also reflect a β-subunit containing the 118 amino acidspredicted from the nucleic acid sequence rather than the 112 found instandard hTSH purified from postmortem human pituitaries.

Transient expression is more convenient than stable integration in theanalysis of a large number of expression vectors. pAV2 and pVARNA nowallow transient expression of hTSH in 293 cells at levels high enough toanalyze protein and glycosylation site structure-function relationships.Previously, the only information about such relationships came fromstudies involving chemical modifications of protein by iodination,nitration, acetylation, and carboxymethylation (Pierce et al. 1981, AnnuRev Biochem, Annual Reviews Inc., Palo Alto, Calif., pp 465-495) orinhibition of glycosylation by tunicamycin (Weintraub, et al. 1980 JBiol Chem 255:5715-5723). The chemical groups could themselves changeprotein conformation irrespective of the alteration in amino acids theyproduce and inhibition of glycosylation affects not only TSH but allcellular glycoproteins. Site-directed mutagenesis of the hCGα cDNA andhuman TSHβ-gene could directly address what regions are important forprotein conformation, subunit combination, receptor binding, biological,activity, and metabolic clearance without introducing chemical groups orunknown changes into the protein structure.

The availability of substantially pure rTSH now makes the diagnosis andtreatment of human thyroid cancer and the determination of the level ofTSH a reality.

Currently, the only available method to diagnose and treat human thyroidcancer involves making patients hypothyroid and allowing their ownendogenous human TSH to rise after several weeks to stimulate the uptakeof ¹³¹I into the cancer. Such stimulation is used as a diagnostic testto localize the tumor by scanning and is subsequently used to treat thecancer by giving large doses of ¹³¹I. All of the diagnostic tests andtherapies depend on high levels of human TSH. However, the technique ofproducing endogenous hypothyroidism has disabling side effects includinglethargy, weakness, cardiac failure, and may also lead to a rapid growthof the tumor over the several week period of treatment. In contrast, ifa desirable form of synthetic human TSH were available, patients couldbe treated while they were euthyroid by giving exogenous injection ofthe TSH. However, presently it is not feasible to give exogenous TSHbecause there is not enough natural product from available humanpituitaries collected at autopsies. Furthermore, even if available, thehuman pituitaries have been found to be contaminated with viruses andthe National Pituitary Agency has forbidden the use of the naturalproduct for any human diagnostic or therapeutic studies. This is truefor all human pituitary hormones including human growth hormone which isnow exclusively marketed as a synthetic product. However, the technologythat was applicable for human growth hormone (a non-glycoprotein) is notat all applicable for human TSH (a glycoprotein hormone of twoglycosylated subunits). As has been described herein supra, only themethodology described herein relating to transfection and properglycosylation of each subunit in mammalian cells produces a desirablebiologically active rTSH material. Moreover, it has been found that thealtered glycosylation pattern that can be achieved with the cells, asdescribed in the methodology of the present invention, produces a longeracting human thyrotropin which is particularly suited for the diagnosisand treatment. of thyroid cancer.

The diagnosis and treatment of thyroid cancer involves first purifyingthe synthetic TSH from large volumes of tissue culture media harvestedfrom approximately ten billion cells over two to four weeks. Using achemically defined medium to reduce protein contaminants as is wellknown in the art, synthetic human TSH, which represents about five toten percent of all the protein secreted into the medium, can beobtained. The human TSH thus obtained is then purified by a combinationof standard techniques including immunoaffinity chromatography, HPLCexclusion chromatography (repeated two to three times) followed bydialysis and concentration by ultrafiltration, lyophilization and thelike. The purified human TSH is then tested in animals to assure itsefficacy as well as freedom from any unexpected toxicity. The syntheticTSH is then tested in patients in clinical trials using different dosesto determine the optimal doses to achieve maximal uptake into the tumorfor both diagnosis and treatment with ¹³¹I. During initial try-outs fordiagnosis, one to two administrations of about 100 μg, while duringtherapy three to six doses of about 100 to 200 μg may be administered,but the optimal dose schedule is determined by the results of theclinical trials. It is noted that all of these procedures areaccomplished while the patient is still euthyroid without producing anyof the disabling side effects of hypothyroidism which are otherwiseencountered in the methods heretofore available.

When the optimal uptake of ¹³¹I has been established, patients may betreated with doses of about 50 to 400 mCi of ¹³¹I and the effect oftherapy assessed by subsequent ¹³¹I diagnostic tests as well asconventional x-rays, CAT scans, measurement of serum thyroglobulin andthe like. Of course, ¹³¹I-labeled rTSH, which is produced by standardmethodology well known in the art, can be appropriately utilized in theprocedures mentioned above.

It is estimated that there will occur about ten thousand new cases ofthyroid cancer in the United States each year and a very largeprevalence of older cases of this cancer require repeated diagnostic andtherapeutic intervention which are currently unsatisfactory.Availability of synthetic human TSH as taught herein, even at a cost of$50.00 to $100.00 per injection will still be a relatively inexpensivepart of the complete evaluation and therapy for this difficult, butcurable cancer.

Another advantage of the product of the present invention is to provideassay components for human thyrotropin using the technique ofradioimmunoassay. Certain immunoassay kits are presently available, butthe reagents therein are again derived from a very short supply ofnatural product. Moreover, the natural product varies greatly dependingon the source of the human pituitaries as well as the degree ofdegradation that occurs during autopsy. This has led to considerable,variation among commercially available kits with disagreements inresults of the TSH testing among various kits. In contrast, the presentinvention, for the first time, provides a virtually unlimited supply ofa stable preparation of synthetic TSH allowing kit manufacturers to havea universal standard preparation and a virtually identical andinexhaustible supply of the reagents. This would allow world wideconsistency of dosage and lead to much needed standardization in themeasurement of human TSH which is vital in the assessment of thyroidfunction in humans. This is accomplished by labeling the rTSH withradioactive iodine. (¹³¹I, ¹²⁵I) or another suitable labeling materialsuch as chemiluminiscent or fluorescent labels and by producingantibodies to the pure product by either polyclonal or monoclonaltechniques which are well known in the art and providing inexhaustiblesupplies of immunoglobulins without significant interferingcross-reactivity with other hormones. The antibodies are then formulatedin classic radioimmunoassay kits which are supplied to the manufacturersto be used in a variety of standard assay methodologies (RIA, IRMA,Sandwich Assays and the like).

There are various other advantages of rTSH. Tests have demonstrated thatit is possible to modify the TSH by expressing the hormone in variouscell lines leading to altered glycosylation patterns. Moreover, usingthe technique of site-directed mutagenesis whereby individual bases inthe DNA are changed, products are obtained with altered biologicfunction such as prolonged or decreased half life, as well ascompetitive antagonists that bind to the TSH receptor and actually blockTSH function. Such competitive antagonists are useful in a novel way totreat diseases such as TSH-induced byperthyroidism as well as Graves'disease which is caused by auto-antibodies to the TSH receptor. Thefunction of these abnormal stimulators would be blocked by thecompetitive antagonist that we have already shown to be active at thecellular level. Moreover, using various long-acting and short-actingpreparations, superagonists can be prepared which would be particularlystimulating to thyroid function, and superantagonists can be preparedwhich would be particularly inhibitory of thyroid function. In thismanner, thyroid function can be controlled in many different types ofdisease of thyroid overactivity or underactivity. It should be notedthat these completely novel approaches are feasible only because of theavailability of the synthetic TSH by the methodology of the presentinvention because, for the reasons mentioned above, the natural productis prohibited from such in vivo use.

It has also been discovered that modifications of the transfectionprocess greatly enhances the amount of TSH production by mammaliancells. For example, instead of using TSH-β gene constructs containingonly the 2nd and 3rd exons (the 2 coding exons), a new construct is madeby adding the first untranslated exon of TSH-β. The inclusion of thisuntranslated TSH-β exon greatly increases TSH production. Without beingbound to any specific theory, it is postulated that the enhanced TSHproduction occurs by increased transcription rate and/or mRNA stability.Moreover, it has been discovered that an excess of the α gene in a ratioof about 3 to 5 times greater than the β gene, yields high rate of TSHproduction (10-50 mg/month), close to commercial scale production.

A standard concentration curve utilizing anti-rTSH antibodies isestablished to determine the amount of TSH in a sample by conventionalimmunological assays.

In summary, a recombinantly made synthetic TSH has been made which, eventhough constitutively distinct from the natural product, possessesfunctional properties similar to the natural product and is useful fordiagnostic as well as therapeutic purposes.

It is understood that the examples and embodiments described herein arefor illustrative purposes only and that various modifications or changesin light thereof will be suggested to persons skilled in the art and areto be included within the spirit and purview of this application andscope of the appended claims. TABLE 1 Immunoassay for Human TSH and itsSubunits in Cell Media from Control and Transfected Cultures. Human TSHβHuman TSH Transfected Construct or Human αRIA (ng/ml) RIA (ng/ml) IRMA(μU/ml) Control (Exp no.) n Mean SEM Mean SEM Mean SEM 293 cells Medium(1, 2) <0.03 <0.03 <0.6 Control (2) 4 0.31  0.01^(a) <0.03 <0.6 Mock (2)4 3.4  0.36^(b) <0.03 <0.6 pVARNA (2) 4 5.0 0.68 <0.03 <0.6 pAV2 (2) 42.1 0.11 <0.03 <0.6 pAV2α (2) 4 1.6 0.31 <0.03 <0.6 pAV2α/pVARNA (2) 417.3 1.0  <0.03 <0.6 PAV2β (2) 4 3.4 0.37 <0.03 <0.6 pAV2β/pVARNA (1) 22.2 0.30 1.5 0.09  2.8 0.30 (2) 4 5.7 0.64 3.3 0.33  6.9 0.64^(a)pAV2α/β/pVARNA (1) 2 4.0 1.5  0.6 0.24  11.2 6.3 (2) 4 6.1 0.54 1.10.09^(d) 15.1 3.6 COS Cells Medium (1, 2) <0.03 <0.03 <0.6 Control (2) 2<0.03 <0.03 <0.6 Mock (2) 2 <0.03 <0.03 <0.6 pAV2α/pVARNA (1) 2 0.060^(b)   <0.03 <0.6 pSV2β (1) 2 <0.03 0.04 0^(d)   <0.6 (2) 2 <0.03 <0.03<0.6 pSV2β/pVARNA (2) 2 <0.03 0.16 0.01^(d) <0.6 pSV2α/β (2) 2 <0.03<0.03 <0.6 pAV2α/pSV2β/pVARNA (1) 2 0.09  0.02^(b) 0.10 0.02^(d) 0.90.06^(a)Various constructs were transfected into either 293 or COS cells in twoseparate experiments. The medium was harvested after 2 days in Exp 1 and3 days in Exp 2. Cell medium was assayed for the human α-subunit, hTSHβsubunit, and hTSH as shown. Human CGα and hTSHβ were labeled α and β inconstruct names.# n, Number of plates transfected. IRMA, 1 μU is immunologicallyequivalent to 0.09 ng NIH I-6 purified hTSH. Medium, Fresh medium beforeapplication to cells. Control, Medium from nontransfected cells. Mock,Medium from cells transfected with a calcium phosphate precipitatelacking DNA.^(a)P < 0.0005 compared to mock, 293 cells^(b)P < 0.0005 compared to pAV2α/pVARNA(2), 293 cells^(c)P < 0.05 compared to pAV2α/β/pVARNA(2), 293 cells^(d)P < 0.005 compared to pAV2β/pVARNA(2), 293 cells.

TABLE 2 Lectin Chromatography of Synthetic and Standard hTSH ColumnUnbound (%) Bound MG (%) Bound-MM 1 WHO STD 0 12 88 2 pAV2α/β/pVARNA 028 72Binding of a synthetic vs. a standard hTSH preparation, described in thelegend to FIG. 4, to concanavalin A-Sepharose, is shown. Results areexpressed as a percentage of the total hTSH immunoactivity eluted fromtwo identical columns. Unbound, Bound-MG, Bound-MM = immunoactivitymeasured in 2 ml column fractions after elution with Tris-bufferedsaline, α-methylglucoside (10 mm), and α-methylmannoside (500 mM),respectively.

1. Substantially pure, biologically active recombinant human thyrotropin(rTSH).
 2. The thyrotropin of claim 1 labeled isotopically with ¹³¹I,¹²⁵I, chemiluminiscently or fluorescently.
 3. The thyrotropin of claim 1produced by recombinant genetic process using constructs with geneelements that enhance thyrotropin production.
 4. A clone comprisingcomplete nucleotide sequence for the expression of the thyrotropin ofclaim 1 in a suitable expression vector.
 5. The clone of claim 4 furthercomprising first untranslated exon of TSH-β.
 6. A method for producingTSH, comprising: (a) allowing expression of TSH by the clone of claim 4in a suitable expression vector; and (b) then recovering substantiallypure TSH by conventional purification and isolation methodology.
 7. Amethod for producing TSH, comprising: (a) allowing expression of TSH bythe clone of claim 5 in a suitable expression vector; and (b) thenrecovering substantially pure TSH by conventional purification andisolation methodology.
 8. A method for producing TSH, comprising: (a)allowing expression of TSH by the clone of claim 4, wherein TSHα isabout 3 to 5 times in excess of TSHβ; (b) then recovering substantiallypure TSH by conventional purification and isolation methodology.
 9. Amethod for producing TSH, comprising: (a) allowing expression of TSH bythe clone of claim 5, wherein TSHα is about 3 to 5 times in excess ofTSHβ; (b) then recovering substantially pure TSH by conventionalpurification and isolation methodology.
 10. A TSH antagonist produced bya mutant of the clone of claim
 4. 11. A TSH agonist produced by a mutantof the clone of claim
 4. 12. A kit comprising containers separatelycontaining: (a) universal standard of substantially pure unlabeled rTSH;(b) substantially pure, labeled rTSH; (c) antibodies against purifiedrTSH; and (d) instructional material describing the use of reagents (a),(b) and (c).
 13. Anti-rTSH antibodies without interferingcross-reactivity with non-TSH hormones.
 14. A method for determining thelevel of TSH in a sample, comprising reacting an aliquot of a sample inwhich the amount of TSH is to be determined with the antibodies of claim13 and comparing the level of antibody reactivity with a predeterminedstandard antibody-rTSH reactivity curve to determine the amount of TSHpresent in said sample.
 15. A method of diagnosing the extent of thyroidcancer, comprising administering rTSH of claim 1 to a patient tomaximize ¹³¹I uptake, and then administering a visualizing dose of ¹³¹Ito said patient; and then visualizing the cancer by standard visualizingmeans.
 16. A method for treating thyroid cancer, comprisingadministering therapeutic regimen of a combination of rTSH of claim 1and ¹³¹I or ¹³¹I-labeled rTSH to a patient afflicted with thyroidcancer.
 17. A method of blocking TSH activity, comprising inhibiting TSHactivity by competitive amount of the antagonist of claim
 10. 18. Amethod of stimulating TSH activity, comprising inducing TSH productionby the agonist of claim 11.